Extraction and concentration method

ABSTRACT

A method for extraction and concentration of antibodies, antigens, bacteria and virus from biological samples. The method also provides a preparation that is suitable for use as a vaccine. The method includes the addition of liquid carboxylic acids and a centrifugation step.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to methods for extraction andconcentration of water and oil soluble compounds from a biologicalsample using liquid carboxylic acids.

[0003] 2. Description of the Related Art

[0004] Salmonella serotype enteritidis (SE) emerged as a major cause ofhuman salmonellosis in the United States throughout the 1980s and 1990s.Of the 360 SE outbreaks with a confirmed source, 279 (82%)) wereassociated with raw or undercooked shell eggs between 1985 and 1998(CDC, 2000). Grade-A shell eggs have been attributed to the humansalmonellosis problem due to the ability of SE to infect ovarian tissuesand to be deposited into the developing egg. It is difficult to detectflocks or individual birds that may be infected with SE and laycontaminated eggs since laying hens seldom show any clinical symptom ofSE infection. Studies indicate that the percentage of infected eggs isextremely low and sporadic with most contaminated eggs having smallnumber of SE (Humphrey, 1994). Culturing both environmental samples andbird tissues does not prove the birds are laying SE infected eggs. Theonly method of directly proving that a flock is laying SE-contaminatedeggs is by culturing the organism from eggs.

[0005] Conventional methods for detection of Salmonella from eggs take5-7 days and are labor-intensive, involving isolation of the organismusing pre-enrichment as well as selective enrichment procedures andserological confirmation tests. Detection of small numbers of SE ininoculated pools of egg contents was successful using direct plating ofincubated egg pools onto agar plates although the more enrichments stepsapplied, the better the sensitivity achieved (Gast, Poultry Science, 72,1611-1614, 1993). Supplementing pools of egg contents with iron in theform of ferrous sulphate and concentrated enrichment broth has beensuggested to improve detection of SE from raw eggs without usingenrichment broth (Gast and Beard, Poultry Sci., Volume 70, 1273-12761991; Cudjoe et al., Int. J. Food Microbiology, Volume 23, 149-158,1994; Gast and Holt, Jour. Food Prot., Volume 61, 107-109, 1998). Morerapid methodologies have been developed using ELISA and PCR techniques(Holt et al., J. Food Prot., Volume 58, 967-972, 1995; Woodward andKirwan, Vet. Rec., Volume 138, 411-413, 1996). In contrast toconventional methods, these tests can detect SE in two days. However,they are not free of drawbacks. The tests involve time-consumingenrichment incubations, exhibit varying degrees of cross-reactions, andboth systems have been known to produce false positive reactions. Thesensitivity of rapid detection methods for SE in eggs was substantiallydecreased due to interference of egg contents (Brigmon et al., PoultryScience, Volume 74, 1232-1236, 1995). Cudjoe et al. (1994, supra) foundthat the more viscous, undiluted mixtures of eggs showed the highestparticle loss compared with diluted samples when immunomagnetic beadswere applied to recover SE from raw eggs.

[0006] U.S. Pat. No. 5,367,054 (Young-Zoon, Nov. 22, 1994) disclosesmethods for isolating and purifying immunoglobulins from eggs whichincludes a first step of extracting a diluted, homogenized egg yolk witha composition containing one or more medium-chain fatty acids, i.e. anyfatty acid having 6-12 carbon atoms, such as caprylic acid, caproicacid, capric acid, and lauric acid while homogenizing the mixture for asecond time.

[0007] U.S. Pat. No. 5,932,250 (Stolle et al., Aug. 3, 1999) disclosesextraction of egg yolk IgY protein fraction using caprylic acid whereegg yolk is diluted 7.5 fold with deionized water and then diluted 1:1with 0.06 M acetate buffer, pH 5. One percent caprylic acid is blendedin and the mixture allowed to stand for 2 hours for separation of theaqueous (bottom) layer and the oil solubles (top layer); withimmunoglobulin in the aqueous layer. The bottom layer is neutralized anddiafiltered and concentrated.

[0008] McLaren et al. (Journal of Immunological Methods, Volume 177,175-184, 1994) disclose extraction of a diluted pool of egg yolk with afinal caprylic acid concentration of 6% (v/v). After the addition ofcaprylic acid, the mixture was stirred for two hours at room temperaturefollowed by centrifugation to obtain a supernatant containingimmunoglobulin.

[0009] While various methods have been disclosed for obtaining proteins,such as immunoglobulins, from biological samples, such as egg yolk,there remains a need in the art for methods for rapid extraction ofproteins, such as immunoglobulins and antigens; and infectious agentssuch as bacteria, from biological samples. The present inventionprovides a method for extracting such proteins and/or infectious agentsfrom biological samples which is fast, provides a concentrated sample,provides increased sensitivity when used in diagnostic testing, and isdifferent from prior art methods and solves some of the problemsassociated with prior art extraction methods.

SUMMARY OF THE INVENTION

[0010] It is therefore an object of the present invention to provide amethod for producing a biological sample for diagnostic testing whichincludes a step of extracting a biological sample with a medium chaincarboxylic acid or mixture of carboxylic acids to obtain an aqueousconcentrated sample.

[0011] Another object of the present invention is to provide anextracted, concentrated sample from a biological material for use in adiagnostic test.

[0012] A still further object of the present invention is to provide anextracted, concentrated, and sterile sample from a biological sample foruse as a vaccine to confer immunity.

[0013] Another object of the present invention is to provide a methodfor concentrating antibodies or antigens in a biological sample fordiagnostic testing wherein a biological sample is extracted with amedium chain carboxylic acid or a carboxylic acid with a carbon numberhigher than 18 which is a liquid and does not damage the material to beextracted and concentrated.

[0014] A further object of the present invention is to provide a methodfor preparing a test sample from an animal egg that includes extractingwhole egg, egg yolk, or egg albumin with a medium chain carboxylic acidor a carboxylic acid with a carbon number higher than 18 which is aliquid and does not damage the material to be extracted and concentratedto form an aqueous sample for diagnostic testing.

[0015] Further objects and advantages of the invention will becomeapparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a photograph of an extracted egg yolk sample aftercentrifugation showing the layers containing oily solubles, esterproducts, and antibody and aqueous solubles.

[0017]FIG. 2 is a photograph of extracted whole egg containing albumin,yolk, and Salmonella enteritidis showing the layers containing oilysolubles, solid reaction products, aqueous solubles, and bacterialpellet.

[0018]FIG. 3 is a photograph of lateral REVEAL™ Salmonella enteritidisflow test panels (Neogen Co., Lansing Mich.) showing a scale of bandintensities in a test panel kit with a control and egg yolk extractscontaining increasing amounts of Salmonella enteritidis.

[0019]FIG. 4 is a photograph of lateral test panels showing detection ofbacteria by yolk (Y) and whole egg contents (W).

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides a method for extracting andconcentrating proteins, such as antigen and antibody; and infectiousagents such as bacteria and virus, from a biological material, such asegg contents, which increases the sensitivity of tests for rapiddetection and provides an inexpensive method for preparing samples fortesting for proteins, such as, for example, immunoglobulins, antigens,and infectious agents such as and without limitation thereto,Salmonella, Newcastle disease, avian leukosis, infectious bursaldisease, adenovirus disease, reovirus, pox, laryngotracheitis, avianinfluenza, Marek's disease, etc. Diagnostic tests include tests such ashemagglutination-inhibition (HI) tests, microagglutination tests (MA),plate agglutination, gel precipitin tests, strip chromatography, ELISA,etc.; or tests based on passing a sample through a beam of light such asspectrophotometry, fluorescence polarization, etc.

[0021] The term “immunoglobulin or “fragment thereof” is intended tomean antibody, especially antibody that is deposited into eggs of avian,reptile, amphibian, or fish during oogenesis. There are three classes ofimmunoglobulins deposited in eggs, IgY, IgA, and IgM. Biologicallyactive fragments of these immunoglobulins are well recognized in the artand include, for example, Fab fragment (having antigen binding site), Fcfragment (the protein domain involved in immune regulation [the fragmentthat crystallizes]), and the Fc′ fragment.

[0022] Medium chain saturated or unsaturated carboxylic acids,specifically fatty acids, including straight chain and branched, havingat least six carbon atoms and up to and including eighteen carbon atomssuch as, for example, caprylic acid, capric acid, oleic acid, isostearicacid, caproic acid, and mixtures thereof. Furthermore any acids ofcarbon numbers higher than 18 that are liquid and do not damage theextracted and concentrated sample, such as protein, bacteria, and virus,are useful.

[0023] The term immune egg is intended to mean eggs from anyegg-producing members of the avian, reptile, amphibian, or fish familywhich have been immunized.

[0024] Avian includes, but is not limited to, poultry and fowl, such aschickens, turkeys, geese, ducks, pheasant, emu, ostrich, etc.

[0025] Reptile includes, but is not limited to, Crocodilia such asalligators, caiman, crocodiles, and garivals, etc.; Chelonia such astortoises and turtles; Squamata such as lizards and snakes, andRhynchocephalia such as tuatara.

[0026] Amphibians include, but are not limited to, frogs, toads, andsalamanders.

[0027] The method of the present invention extracts and concentratesproteins such as antibodies, antigens; and bacteria or viruses, such asSalmonella enteritidis, for example, in an aqueous medium to providesamples for use in conventional or automated assays. The method providesclear, particle free samples (See FIGS. 1 and 2) that can be applied toconventional serological tests and used in methods where measurementsare based on beams of light. The method is fast, employing a mixing stepand a centrifugation step. The method removes organisms such asSalmonella enteritidis from samples to provide a preparation that issuitable for use as a vaccine, in some instances such as with Salmonellaenteritidis, the preparation should be sterile (FIG. 2). Samples can bepooled which saves time and increases scanning range. The extractedsamples can be further processed for concentration by precipitationmethods to increase sensitivity. Bulk extraction and concentration isalso an option. The method provides samples that can be used directly orwith minimal processing on chromatographic columns to detect subgroupsof antibody and their amounts.

[0028] Extraction of antibody from egg yolks or blended whole eggcontents for assay of specific antibody against disease is oneapplication of the present invention. Eggs are a quick source ofantibodies and their collection does not interfere with egg lay orinvolve contact with the animal as does bleeding for serum testing. Themethod is also applied for detection of antigen to determine infectionby a pathogen. Furthermore, oil emulsion vaccines can be separated intooil and aqueous phase to determine antigen amount and its relevance topotency before purchase. The extract can also be used for antibodyrecovery and purification in high yield since the antibody extract onlyrequires two steps from egg yolk and only requires a dialysis step andone pass through a thiophilic chromatography column. Furthermore, theextracts are sterile and can be injected into embryos to establish earlyprotection in hatched chicks using the low viscosity soluble eggcomponents compatible for embryos.

[0029] The method of the present invention includes extracting abiological sample such as whole egg, egg yolk, egg albumin, bacteria ingrowth mediums, and infected tissues, for example, with a medium chainsaturated or unsaturated carboxylic acid, such as for example, anycarboxylic acid that is a liquid near or at room temperature, caprylicacid, capric acid, oleic acid, isostearic acid, or mixtures thereof orwith at least one carboxylic acid with a carbon number higher than 18which is a liquid and does not damage the material to be extracted andconcentrated. Ratios of organic acid and samples are used that providemaximum yield of soluble product and that provide a viscosity that willallow a thorough mixing. Some ratios, 1:1 for example, will result in aviscous admix since the esters formed are surfactants and will emulsifythe aqueous product causing the generation of new surfaces which resultsin friction (viscosity). Addition of oily acid or sample will lowerviscosity in samples that emulsify. Very strong mixing should be avoidedsince this may generate strong water-in-oil emulsions and the aqueousproduct can not be centrifuged out of these. A ratio of about two partsbiological sample to about 1 part acid (v:v or w:w) is preferred formost mixtures used in small (approximately ≦2 ml) centrifuge tubes. Thesample with acid is mixed until the sample is viscous, at least about 1minute, using any conventional means for mixing such as, for example,stirring, vortexing, blending, mechanical or manual shaking, etc.Blenders and emulsifying machines should be used at low speed to preventforming an emulsion. The sample is then centrifuged for about 5 to 10minutes at about 10,000×g to about 16,000×g. For bacterial and viralcomponent recovery, the sample is centrifuged at about 16,000×g orhigher to pellet antigens or about 1000×g to about 10,000×g to suspendantigen. One of ordinary skill in the art could readily determinecentrifuge speed based on the density of the antigen in the sample. Thebottom aqueous layer is sampled for assay using any conventional meansfor sampling such as, for example, syringe, pipet, cutting bottom oftube, etc.

[0030] The following examples are intended to further illustrate theinvention and are not intended to limit the scope of the invention whichis defined by the claims. Chicken eggs are used as a model system forthe present invention.

EXAMPLE 1

[0031] Single-comb white leghorn hens, 26-32 weeks old, from theUSDA-Agricultural Research Service Southeast Poultry Research Laboratory(Athens, Ga.) specific-pathogen-free flock (SPF) were reared in diseasecontainment buildings with appropriate diet and husbandry. They werehoused in individual laying cages and grouped in separate rooms fordifferent treatments. They were fed water and feed ad libitum.

[0032] One ml of blood was drawn from the wing vein for serum collectionimmediately before challenge of hens and at weekly intervals.

[0033] Eggs were collected daily and identified individually for eachhen. Egg yolks were separated from the albumin by cracking the egg,dropping most of the albumin and rolling the intact yolk over into a 50ml centrifuge tube. Residual albumin was removed with a syringe when theyolk was at the lip of the tube. The whole yolk was mixed to assure evenantibody distribution before sampling.

[0034] For whole egg contents, eggs were cracked and the contentsemptied into a narrow neck 250 ml blender cup (Waring blender, DynamicsCorp. of America, New Hartford, Conn.). They were blended at the lowsetting with on-off switching, as needed, to prevent formation of avortex which forms bubbles and ultra fine particles that interfere withthe fluorescence polarization (FP) assay. This process usually takesabout 4 seconds or less.

[0035] For extraction of yolk and whole egg contents, about a one mlsample and about a 0.5 ml sample of a mixture of 2 parts oleic acid and1 part caprylic acid were mixed with an applicator stick in a 1.7 mlEppendorf centrifuge tube for about 2 minutes or until the mixturebecame viscous. The tube is then centrifuged at about 14,000×g for aboutten minutes. The clear solution at the bottom of the tube (about 0.2 mlfor yolk and about 0.5 ml for whole egg contents) was then sampled(about 20 μl unchanged or undiluted in about 1 ml of phosphate bufferedsaline (PBS)) for the FP assay. The acids react with the activehydrogens in the sample forming water which solubilized the antibody.This soluble antibody was used for assay.

[0036] For the fluorescence assay, instrumentation and fluorescenceantigen (tracer) was developed by Diachemix Corp. (Grayslake, Ill.). Onedisposable 10×75 mm glass tube (Fisher Scientific, Pittsburgh, Pa.)containing about 1 ml of phosphate buffered saline (PBS) is required foreach assay. The instrumentation is equilibrated one time for temperatureand antigen spin in one ml of PBS for a baseline value. For antibodytesting, about 20 μl of each sample is mixed in about 1 ml of PBS pertube and equilibrated (blanked) on the machine for a baseline. Then thetube is removed and about 10 μl of tracer (fluorescent antigen) isadmixed gently. The tube is replaced and the polarized beam of themachine is allowed to sweep the sample 25 times to reach an endpointvalue. Each antibody determination takes less than three minutes.Samples (PBS and sample) can be mixed ahead of time before the tracer isadded. FP positive values are those two standard deviations above themean FP values for the negative control samples.

[0037] Two experiments, each with two replicate trials were done.Experiment one tested serum and yolk samples while experiment two testedwhole egg contents. Trial one of experiment one used 26 week old hens,six uninoculated controls and three groups of ten receiving about a 1 mloral administration into the crop containing about 10⁶ colony formingunits (CFU) of a phage type 13a strain of Salmonella enteritidis, about10⁸ CFU of the same strain, or about 1.2×10⁸ CFU of Salmonellatyphimurium. Salmonella strains were regenerated from frozen stocks by37° C. incubation in tryptone soya broth (Oxoid USA, Ogdensburg, N.Y.)overnight. Cell density was adjusted to about a 1 ml dose in the broth.

[0038] For trial two of experiment one, the same treatments were givento about 32 week old hens.

[0039] The eggs from trial one were collected, as described above, andthe contents of one egg from each individual hen at about 22-24 dayspost challenge were blended and tested for antibodies in the extractionor untreated supernatants obtained by centrifugation (about 14,000×g forabout 10 minutes) or untreated supernatants obtained from overnightsettling. The three samples were tested by FP, microagglutination (MA),Pullorum test (PT) where possible, or gel precipitation (AGPT) forantibodies against Salmonella enteritidis.

[0040] Eggs from trial two were pooled (2-5 eggs) from individual hensthrough the 4^(th) week after challenge and their contents blended. Thepools were tested by FP only. Samples of the pools tested were extractedwith the acid mixture as described above.

[0041] The results of trials one and two are shown below in Tables 1 and2, respectively. Hens of the non-infected control groups were negativefor Salmonella enteritidis by all tests of both trails. The 10⁶ CFUgroups of both trial were serologically positive by FP from about 66.7to about 80 percent over 14 to 35 days post infection. Yolks of thesehens were SE antibody positive by FP from about 37.5 to about 100%. ThePT, MA and AGPT tests all indicated about 83.3% positive at about 35days post infection. In both trials of Salmonella typhimurium infectedhens FP disclosed a range of about 30 to 60% positives in the sera. Inthe yolks the range was about 30-90%. For the PT, MA, and AGPT tests atabout 35 days post infection, the positives were about 28.6%, 42.9% and14.3%, respectively.

[0042] In experiment two, hatchmate hens in separate disease containmentrooms were challenged with Salmonella enteritidis by either the oral,intravenous (IV) or aerosol route. Aerosol challenge was delivered byspraying in the face with about a 10 ml volume containing about 10⁹ CFU.IV challenge was with about a 0.5 ml dose of about 10⁷ or about 10⁵ CFUinto the wing vein. Oral challenge was by gavage of about a 1 ml volumeinto the crop. The samples were collected as described above forexperiment one.

[0043] For experiment 2, trial 1, egg samples of all non-infected henswere negative by all applicable tests. The egg samples of the IVinfected group were all positive in all sample treatment groups by alltests. In the aerosol infected groups FP detected the most positiveswith about 93.3%, 73.3%, and 73.3% for the extracts, supernatants fromcentrifugation, and from settling, respectively. Positives by FP in theorally infected group were about 61.1%, 88.9%, and 61.1% forcorresponding treatment groups. With the exception of the PT on theextract of the orally infected group (about 88.9%) values from theconventional tests ranged from about 0% to about 60% for aerosol andoral groups (Table 3 below).

[0044] In trial 2, samples of the nonimmune groups were negative for theblended only group. The extract and centrifuged supernatant controlgroups showed about 6.25% positives. The IV challenged groups were 100%positive. Positives for the extracts and blended only samples were thesame at about 83.3% and 85.7% for aerosol and oral challenges. Among thesupernatant samples, positives were about 66.7% for aerosol and about64.3% for oral challenges (Table 4 below). TABLE 1 Percent positivesdetected by Fluorescence polarization in serum and yolk extracts. DaysPost Infection 14 21 28 Controls serum 00.0 00.0 00.0 yolk 00.0 00.000.0 10⁶ CFU serum 77.8 66.7 77.8 yolk 66.7 55.6 100.0  10⁸ CFU serum100.0  100.0  100.0  yolk 100.0  86.7 100.0  10⁸ CFU ^(A)ST serum 40.050.0 30.0 yolk — 30.0 90.0

[0045] TABLE 2 Percent Positives detected by Fluorescence polarizationin serum and yolk extracts. Days Post Infection 21 28 35 Controls serum00.0 00.0 00.0 yolk 00.0 00.0 00.0 ^(A)yolk PT — — 00.0 ^(B)yolk MA — —00.0 ^(C)yolk AGPT — — 00.0 10⁶ CFU serum 80.0 77.8 77.8 yolk 37.5 87.566.7 ^(A)yolk PT — — 83.3 ^(B)yolk MA — — 83.3 ^(C)yolk AGPT — — 83.010⁶ CFU serum 90.0 80.0 90.0 yolk 85.7 100.0  100.0  ^(A)yolk PT — —90.0 ^(B)yolk MA — — 80.0 ^(C)yolk AGPT — — 90.0 10⁸ CFU ^(D)ST serum60.0 30.0 67.7 yolk 00.0 67.7 42.9 ^(A)yolk PT — — 28.6 ^(B)yolk MA — —42.9 ^(C)yolk AGPT — — 14.3

[0046] TABLE 3 Percent positives detected 24 days post infection inblended whole egg contents by fluorescence polarization and conventionaltests. Route of Infection Sample treatment Aerosol Intravenous Oral AcidExtracts FP 93.3 100.0  61.1 Control 00.0 00.0 00.0 MA 13.3 100.0  44.4Control 00.0 00.0 00.0 PT 60.0 100.0  88.9 Control 00.0 00.0 00.0 AGPT20.0 100.0  33.3 Centrif. Sup. FP 73.3 100.0  88.9 Control 00.0 00.000.0 MA 06.7 100.0  22.2 Control 00.0 00.0 00.0 PT NA NA NA AGPT 00.0100.0  11.1 Control 00.0 00.0 00.0 Settling Sup. FP 73.3 100.0  61.1Control 00.0 00.0 00.0 MA 00.0 100.0  16.7 Control 00.0 00.0 00.0 PT NANA NA AGPT 00.0 100.0  11.1 Control 00.0 00.0 00.0

[0047] TABLE 4 Percent positives detected by fluorescence polarizationin individual blended whole egg pools of hens infected by differentroutes. Four Weeks post infection. Route of Infection Sample treatmentAerosol Intravenous Oral Acid Extracts 83.3  100.0  85.7  Control 06.2506.25 06.25 Centrif. Sup 66.7  100.0  64.3  Control 06.25 06.25 06.25Blended only 83.3  100.0  85.7  Control 00.0  00.0  00.0 

EXAMPLE 2

[0048] This example demonstrates the use of fatty acid extraction of thepresent invention to detect the presence of Salmonella in eggs.Salmonella enteritidis (SE) was obtained from stocks kept at theSoutheast Poultry Research Laboratory (SEPRL, Athens, Ga.). SE wasselected for resistance to rifampicin (Seo et al., J. Food Prot., Volume63, 545-548, 2000). Purity of the cultures was confirmed usingserotyping (Difco Laboratories, Detroit, Mich.). Cultures were grown intryptic soy broth (TSB; Difco) and viable counts were obtained byplating 10-fold serial dilutions of broth cultures on nutrient agar(Difco) and incubating plates at 37° C. overnight. Whenever necessary,cultures were diluted in phosphate buffered saline (PBS, pH 7.2) asneeded and counts were made as above.

[0049] A lateral flow panel test (Neogen Co., REVEAL™ Salmonellaenteritidis, Lansing, Mich.) (FIG. 1) was used to determine the presenceof Salmonella in about a 100 μl portion of a sample that was placed intothe round sample port of the test device. The test panel is based onchromatography. This initiated lateral flow through a reagent zonecontaining specific anti-Salmonella enteritidis antibodies conjugated tocolloidal gold particles. The antibody used in the device was developedat the SEPRL and disclosed in a previous study (Holt et al., J. FoodProt., Volume 58, 967-972, 1995). Test results were interpreted aspositive or negative, scored on a scale from 0 to 4, after 20 minutesfrom the addition of the sample culture (FIG. 1).

[0050] In order to test the detection limit of the assay, pure culturesof SE were grown in TSB and tested at concentrations between 10⁰ to 10⁸cells/ml.

[0051] To determine the minimum number of SE needed for generating apositive result, eggs were spiked with a known number of SE. Eggs werecollected from the SEPRL specific-pathogen-free flock and were soaked inabout 70% alcohol to disinfect the shells before they were broken. Theegg contents were pooled and homogenized for about 1 minute in aStomacher. About 10 mls of egg contents were allocated into 15 ml testtubes and mixed with about 100 μl of different 10-fold SE dilutions togenerate samples with known number or organisms. For negative controls,about 100 μl of PBS was mixed with about 10 ml of egg contents.

[0052] In order to enhance the ability to detect SE, egg contents wereeither diluted with PBS, extracted with a mixture of oleic and caprylicacid, or enriched by culturing in broth for about 24 hours at 37° C. Fordilution, egg contents with different numbers of SE were diluted withPBS at about 1:2, 1:4, and 1:10 and about 100 μl of each sample wasadsorbed to a test panel to find the optimum running ability of thesample and detection limit.

[0053] For extraction, one ml of whole egg with various concentrationsof SE and 0.5 ml of a mixture of about 2 parts oleic acid and one partcaprylic acid were stirred together with an applicator stick in a 1.7 mlEppendorf centrifuge tube to allow the acid to react and form an esterwith reactive hydroxyl groups. The reaction forms water as a by-productand at the same time breaks up the structure of the egg contents whichreleases the bacteria and aqueous solubles. The reaction is nearcompletion when the mixture becomes viscous, in approximately 1-2minutes at room temperature. The tube is then centrifuged for about 5minutes at about 10,000×g in an Eppendorf tabletop centrifuge.Centrifugation forms an oily layer with yellow carotenes and oilsolubles on the top, a soapy hard layer of esters in the middle, and aclear aqueous layer with bacteria and soluble protein on the bottom(FIG. 2). The aqueous layer which contains antigen and bacteria wasremoved with a 1 ml syringe and about 100 μl of the solution was appliedto a test panel.

[0054] For enrichment, each homogenized egg sample containing a known SEconcentration was mixed with TSB at various ratios between egg contentsand enrichment broth. After about a 24 hour incubation at about 37° C.,about 100 μl of each broth culture was adsorbed onto a test panel andthe results were determined after about 20 minutes.

[0055] The minimum concentration of SE to generate a positive band onthe test panel was approximately 10⁷ cells/ml in pure culture and nocross-reactivity was detected with other salmonella serovars. Onevariable which is crucial for the success of the test panel is theability of the sample to be effectively wicked down the membrane so thatthe organism or antigen present in the sample can interact with thedetection antibodies. Whole egg does not effectively transit the paneldue to the viscous nature of the sample (Table 5 below). Progressivelydiluting the egg with PBS reduces the viscosity of egg contents andallows the more effective wicking of sample. About a 1:10 dilution ofsample with PBS works best. However, the intensity of the positive bandappeared weaker as the dilution was increased resulting in lowsensitivity to about 10⁸ cells/ml. Similar drawbacks of immunoassayswere reported in a previous study (Brigmon et al., 1995; supra) whenthey used ELISA to detect SE in eggs. The sensitivity of the ELISAsystem was decreased to 10⁷ cells/ml when SE was mixed with 10%homogenized whole eggs while SE was detected at a minimum concentrationof 10⁴ cells/ml in pure culture. This suggests that the albumin andlipids in yolk may act as blocking agents inhibiting antibody binding inthe ELISA. Extraction of bacterial antigen out of egg contents usingfatty acid provided excellent running capability and stronger positivesignal on the test panel kit without sacrificing the sensitivity. Thedetection limit of the test kit increased to about 10⁶ cells/ml in wholeegg contents using fatty acid extraction (Table 6). Egg pools inoculatedwith 1-5 cells/ml of SE were detected as SE-positive after 24 hoursincubation at 37° C. when the direct antigen extraction method was usedwith the test panel (Table 7). This technique could lead to relativelyrapid and inexpensive detection of SE in whole eggs by saving enrichmentmedia and reducing incubation time. TABLE 5 Direct detection of SE fromwhole egg contents using dilution technique with PBS and antigenextraction with a fatty acid mixture. Egg:PBS Egg:PBS Egg:PBS Egg:acidWhole egg (1:2) (1:4) (1:10) (2:1) Band −^(A) ++ ++ + +++ intensitysample poor not good good very good very good Running Controls — — — — —

[0056] TABLE 6 Detection limit of direct detection of SE from whole eggcontents using dilution technique with PBS and antigen extraction withfatty acid mixture. METHODS Number of Dilution Fatty Acid cells (cFU/ml)Technique Extraction 10⁸ +++ +++ 10⁷ + +++ 10⁶ − ++ 10⁵ − −+

[0057] TABLE 7 Direct detection of SE from whole egg contents usingdilution technique with PBS and antigen extraction with fatty acidmixture. Methods Fatty Acid Samples Dilution Technique MixtureExtraction Negative control −(3/3)      −(3/3)  Inoculated Eggs +(10/10)+++(10/10)

EXAMPLE 3

[0058] Samples were prepared using fatty acid extraction for bacterialdetection using a lateral panel as in example 2 with specific Salmonellaenteritidis antibodies conjugated with gold particles. Eggs werecontaminated as in Example 2. Egg yolk or whole egg contents wereextracted as described above in Example 2 using oleic acid.Approximately 100 μl of the aqueous layer was removed and applied to thepanel well. The reaction was complete within about one to two minutes(FIG. 4). No sensitivity is lost since there is no dilution of thesample as would occur if the albumin or yolk is diluted with saline andadded directly to the test strip. Whole egg contents and albumin aremore readily extracted than yolk.

EXAMPLE 4

[0059] Fatty acids were tested for extraction efficiency of Salmonellaenteritidis antibodies from whole eggs. Hens were infected by IV (about10⁷ or about 10⁵ CFU in about a 0.5 ml dose in the wing vein) with SE asdescribed in Example 1, experiment 2. Whole egg contents were preparedas in Example 1 and were directly, i.e. no dilution, mixed approximately2:1 with caproic, caprylic, capric, oleic, or isostearic acid asdescribed in Example 2, allowed to react until sample became viscous,approximately 1-2 minutes, and centrifuged for about 10 minutes at about16,000×g. The clear aqueous layer was sampled, as described in example1, for fluorescence polarization assay.

[0060] The results show that caprylic acid by itself is no differentfrom the untreated immune control. Capric, oleic, and isostearic acidsyielded nearly the same antibody titers at about 11 to about 16 pointsabove the untreated immune control. Caproic acid did not yield a meanantibody titer above the non-immune control. TABLE 8 Homologousfluorescence polarization titers against Salmonella enteritidis fromvarious fatty acid extractions of whole egg contents. Cummulativenonimmune Positive samples pos. Diff. over Organic Acid control 1 2 3 4mean neg. control no treatment 95.8 163.1 146.8 193.4 193.9 174.3 78.5supernatant caproic (C6) 94.8 86.9 94.1 97.8 96.8 93.9 −0.9 caprylic(C8) 90.4 162.1 139.6 187.2 192.0 170.2 79.8 capric (C10) 94.1 — 158.5207.8 200.6 189.0 95.0 oleic (C18) 92.7 171.4 153.4 206.0 197.3 182.089.3 Isostearic (C18) 91.5 172.4 150.3 206.4 200.6 182.4 90.9

[0061] The foregoing detailed description is for the purpose ofillustration. Such detail is solely for that purpose and those skilledin the art can make variations without departing from the spirit andscope of the invention.

What is claimed is:
 1. A method of extraction for a biological samplecomprising: (a) adding at least one liquid carboxylic acid to a liquidsample to form a first composition, (b) mixing said first composition toform a second composition that contains an aqueous soluble product, (c)centrifuging said second composition to form an aqueous layer and anoily layer, and (d) harvesting said aqueous layer to produce aconcentrated sample selected from the group consisting of proteins,bacteria, virus, and mixtures thereof.
 2. The method of claim 1 whereinsaid at least one liquid carboxylic acid is selected from the groupconsisting of caprylic acid, capric acid, oleic acid, isostearic acid,caproic acid and mixtures thereof.
 3. The method of claim 1 wherein saidat least one liquid carboxylic acid is a mixture of two carboxylicacids.
 4. The method of claim 3 wherein said mixture of two carboxylicacids is selected from the group consisting of caprylic acid, capricacid, oleic acid, isostearic acid, and caproic acid.
 5. A method forextraction of a biological sample consisting essentially of: (a) addingat least one liquid carboxylic acid to a biological sample to form afirst composition, (b) mixing said first composition to form a secondcomposition that contains an aqueous soluble product, (c) centrifugingsaid second composition to form an aqueous layer and an oily layer, and(d) harvesting said aqueous layer to produce a concentrated sampleselected from the group consisting of proteins, bacteria, virus, andmixtures thereof.
 6. The method of claim 5 wherein said at least onecarboxylic acid is selected from the group consisting of caprylic acid,capric acid, oleic acid, isostearic acid, caproic acid, and mixturesthereof.
 7. The method of claim 5 wherein said at least one liquidcarboxylic acid is a mixture of two liquid carboxylic acids.
 8. Themethod of claim 7 wherein said mixture of two liquid carboxylic acids isselected from the group consisting of caprylic acid, capric acid, oleicacid, isostearic acid, and caproic acid.
 9. A method for obtainingpurified antibodies from a biological sample comprising: a. adding atleast one liquid carboxylic acid to a sample to form a firstcomposition, b. mixing said first composition to form a secondcomposition that contains an aqueous soluble product, c. centrifugingsaid second composition to form an aqueous layer and an oily layer, d.harvesting said aqueous layer to produce an extracted and concentratedantibody sample, e. dialyzing said antibody sample, and f. passing saiddialysate over a chromatographic column to obtain a purified antibodypreparation.
 10. The method of claim 9 wherein said at least one liquidcarboxylic acid is selected from the group consisting of caprylic acid,capric acid, oleic acid, isostearic acid, caproic acid, and mixturesthereof.
 11. The method of claim 9 wherein said at least one liquidcarboxylic acid is a mixture of two carboxylic acids.
 12. The method ofclaim 11 wherein said mixture of two carboxylic acids is selected fromthe group consisting of caprylic acid, capric acid, oleic acid,isostearic acid, and caproic acid.
 13. A method for obtaining purifiedantibodies from a biological sample consisting essentially of:. a.adding at least one liquid carboxylic acid to a sample to form a firstcomposition, b. mixing said first composition to form a secondcomposition that contains an aqueous soluble product, c. centrifugingsaid second composition to form an aqueous layer and an oily layer, d.harvesting said aqueous layer to produce an extracted and concentratedantibody sample, e. dialyzing said antibody sample, and f. passing saiddialysate over a chromatographic column to obtain a purified antibodypreparation.
 14. The method of claim 13 wherein said at least one liquidcarboxylic acid is selected from the group consisting of caprylic acid,capric acid, oleic acid, isostearic acid, caproic acid, and mixturesthereof.
 15. The method of claim 13 wherein said at least one liquidcarboxylic acid is a mixture of two carboxylic acids.
 16. The method ofclaim 15 wherein said mixture of two carboxylic acids is selected fromthe group consisting of caprylic acid, capric acid, oleic acid,isostearic acid, and caproic acid.
 17. A method for detecting thepresence of an infectious agent in a biological sample comprising: a.adding at least one liquid carboxylic acid to a sample to form a firstcomposition, b. mixing said first composition to form a secondcomposition that contains an aqueous soluble product, c. centrifugingsaid second composition to form an aqueous layer and an oily layer, d.harvesting said aqueous layer to produce an extracted and concentratedantibody sample, e. adding said harvested aqueous layer to an assay todetermine the presence of an infectious agent.
 18. The method of claim17 wherein said at least one liquid carboxylic acid is selected from thegroup consisting of caprylic acid, capric acid, oleic acid, isostearicacid, caproic acid, and mixtures thereof.
 19. The method of claim 17wherein said at least one liquid carboxylic acid is a mixture of twocarboxylic acids.
 20. The method of claim 19 wherein said mixture of twocarboxylic acids is selected from the group consisting of caprylic acid,capric acid, oleic acid, isostearic acid, and caproic acid.
 21. Themethod of claim 19 wherein said harvested aqueous layer is applied to atest strip containing antibodies directed to said infectious agent. 22.A method for detecting the presence of an infectious agent in abiological sample consisting essentially of: a. adding at least oneliquid carboxylic acid to a sample to form a first composition, b.mixing said first composition to form a second composition that containsan aqueous soluble product, c. centrifuging said second composition toform an aqueous layer and an oily layer, d. harvesting said aqueouslayer to produce an extracted and concentrated antibody sample, e.adding said harvested aqueous layer to an assay to determine thepresence of an infectious agent.
 23. The method of claim 22 wherein saidat least one liquid carboxylic acid is selected from the groupconsisting of caprylic acid, capric acid, oleic acid, isostearic acid,caproic acid, and mixtures thereof.
 24. The method of claim 22 whereinsaid at least one liquid carboxylic acid is a mixture of two carboxylicacids.
 25. The method of claim 24 wherein said mixture of two carboxylicacids is selected from the group consisting of caprylic acid, capricacid, oleic acid, isostearic acid, and caproic acid.
 26. The method ofclaim 22 wherein said harvested aqueous layer is applied to a test stripcontaining antibodies directed to said infectious agent.
 27. Anextracted concentrated sample produced by the method of claim
 1. 28. Anextracted concentrated sample produced by the method of claim
 5. 29. Anextracted concentrated antibody produced by the method of claim
 9. 30.An extracted concentrated antibody produced by the method of claim 13.31. An extracted, concentrated infectious agent produced by the methodof claim
 17. 32. An extracted, concentrated infectious agent produced bythe method of claim 22.